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      Adenoviral Producer Cells

      review-article
      , *
      Viruses
      Molecular Diversity Preservation International (MDPI)
      producer cell lines, adenovirus, Ad vectors, RCA

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          Abstract

          Adenovirus (Ad) vectors, in particular those of the serotype 5, are highly attractive for a wide range of gene therapy, vaccine and virotherapy applications (as discussed in further detail in this issue). Wild type Ad5 virus can replicate in numerous tissue types but to use Ad vectors for therapeutic purposes the viral genome requires modification. In particular, if the viral genome is modified in such a way that the viral life cycle is interfered with, a specific producer cell line is required to provide trans-complementation to overcome the modification and allow viral production. This can occur in two ways; use of a producer cell line that contains specific adenoviral sequences incorporated into the cell genome to trans-complement, or use of a producer cell line that naturally complements for the modified Ad vector genome. This review concentrates on producer cell lines that complement non-replicating adenoviral vectors, starting with the historical HEK293 cell line developed in 1977 for first generation Ad vectors. In addition the problem of replication-competent adenovirus (RCA) contamination in viral preparations from HEK293 cells is addressed leading to the development of alternate cell lines. Furthermore novel cell lines for more complex Ad vectors and alternate serotype Ad vectors are discussed.

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          Most cited references110

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          Characteristics of a human cell line transformed by DNA from human adenovirus type 5.

          Human embryonic kidney cells have been transformed by exposing cells to sheared fragments of adenovirus type 5 DNA. The transformed cells (designated 293 cells) exhibited many of the characteristics of transformation including the elaboration of a virus-specific tumour antigen. Analysis of the polypeptides synthesized in the 293 cells by labelling with 35S-methionine and SDS PAGE showed a variable pattern of synthesis, different in a number of respects from that seen in otheruman cells. On labelling the surface of cells by lactoperoxidase catalysed radio-iodination, the absence of a labelled polypeptide analogous to the 250 K (LETS) glycoprotein was noted. Hybridization of labelled cellular RNA with restriction fragments of adenovirus type 5 DNA indicated transcription of a portion of the adenovirus genome at the conventional left hand end.
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            A simplified system for generating recombinant adenoviruses.

            Recombinant adenoviruses provide a versatile system for gene expression studies and therapeutic applications. We report herein a strategy that simplifies the generation and production of such viruses. A recombinant adenoviral plasmid is generated with a minimum of enzymatic manipulations, using homologous recombination in bacteria rather than in eukaryotic cells. After transfections of such plasmids into a mammalian packaging cell line, viral production is conveniently followed with the aid of green fluorescent protein, encoded by a gene incorporated into the viral backbone. Homogeneous viruses can be obtained from this procedure without plaque purification. This system should expedite the process of generating and testing recombinant adenoviruses for a variety of purposes.
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              CD46 is a cellular receptor for group B adenoviruses.

              Group B adenoviruses, a subgenus of human Adenoviridae, are associated with a variety of often-fatal illnesses in immunocompromised individuals, including bone marrow transplant recipients and cancer and AIDS patients. Recently, group B adenovirus derivatives have gained interest as attractive gene therapy vectors because they can transduce target tissues, such as hematopoietic stem cells, dendritic cells and malignant tumor cells, that are refractory to infection by commonly used adenoviral vectors. Whereas many adenoviruses infect cells through the coxsackievirus and adenovirus receptor (CAR), group B adenoviruses use an alternate, as-yet-unidentified cellular attachment receptor. Using mass spectrometric analysis of proteins interacting with a group B fiber, we identified human CD46 as a cellular attachment receptor for most group B adenoviruses. We show that ectopic expression of human CD46 rendered nonhuman cells susceptible to infection with group B viruses in vitro and in vivo. In addition, both siRNA-mediated knockdown of CD46 and a soluble form of CD46 blocked infection of human cell lines and primary human cells. The discovery that group B adenoviruses use CD46, a ubiquitously expressed complement regulatory protein, as a cellular attachment receptor elucidates the diverse clinical manifestation of group B virus infections, and bears directly on the application of these vectors for gene therapy.
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                Author and article information

                Journal
                Viruses
                Viruses
                Molecular Diversity Preservation International (MDPI)
                1999-4915
                August 2010
                16 August 2010
                : 2
                : 8
                : 1681-1703
                Affiliations
                VectorLogics, Inc., 550 11th Street South, Birmingham, AL35294, USA; E-Mail: ikovesdi@ 123456vectorlogics.com
                Author notes
                [* ]Author to whom correspondence should be addressed; E-Mail: shedley@ 123456vectorlogics.com ; Tel.: +1-205-933-8378; Fax: +1-205-933-5836.
                Article
                viruses-02-01681
                10.3390/v2081681
                3185730
                21994701
                494d7dd1-7794-44b1-8e76-ccc96cde51ea
                © 2010 by the authors; licensee MDPI, Basel, Switzerland.

                This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license ( http://creativecommons.org/licenses/by/3.0/).

                History
                : 16 June 2010
                : 30 July 2010
                : 13 August 2010
                Categories
                Review

                Microbiology & Virology
                rca,adenovirus,ad vectors,producer cell lines
                Microbiology & Virology
                rca, adenovirus, ad vectors, producer cell lines

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